Breast Cancer Risk Is Associated with the Genes Encoding the DNA Double-Strand Break Repair Mre11/Rad50/Nbs1 Complex

2024

Huan-Ming Hsu,1,2,3 Hui-Chun Wang,2 Sou-Tong Chen,6 Giu-Cheng Hsu,4 Chen-Yang Shen,2,5 and Jyh-Cherng Yu3 1Graduate Institute of Medical Sciences, National Defense Medical Center; 2Institute of Biomedical Sciences, Academia Sinica; Departments of 3Surgery and 4Radiology, Tri-Service General Hospital; 5Life Science Library, Academia Sinica, Taipei, Taiwan; and 6Department of Surgery, Changhua Christian Hospital, Changhua, Taiwan

Abstract

The evolutionarily conserved Mre11-Rad50-Nbs1 observations that (a) one single-nucleotide polymor- (MRN) complex, consisting of proteins encoded by phism in Nbs1 was significantly associated with breast the genes Mre11, Rad50, and Nbs1, was recently shown cancer risk, and a trend toward an increased risk of to play a crucial role in DNA double-strand break developing breastcancer was found in women harbor- (DSB) repair by recruiting the nuclear protein kinase ing a greater number of putative high-risk genotypes of ataxia telangiectasia mutated to DSB sites, leading to MRN genes (an adjusted odds ratio of 1.25 for each activation of this DNA repair network. Given the fact additional putative high-risk genotype; 95% confidence that carriers of defective mutation and polymorphic interval, 1.10-1.44); (b) this association between risk and variants of ataxia telangiectasia mutated are athigher the number of putative high-risk genotypes was risk of developing breastcancer, we hypothesizeda stronger and more significant in women thought to be role of the MRN genes in determining breast cancer more susceptible to estrogen, i.e., those with no history susceptibility. This hypothesis was examined both in a of full-term pregnancy, those older (z26 years of age) at case control study of 559 breast cancer patients and first full-term pregnancy, or those having had fewer 1,125 healthy women of single-nucleotide polymor- (<2) full-term pregnancies; the risk effect conferred by phisms in Mre11, Rad50, and Nbs1 and by the in vivo harboring a higher number of high-risk genotypes of detection of binding between Mre11 and BRCA1, MRN genes was more significantin women withouta encoded by the breast cancer susceptibility gene history of breast feeding; and (c) Mre11 and BRCA1 BRCA1. We were also interested in defining whether were shown to form a complex in vivo, and this any association between MRN genes and breast cancer interaction was increased by irradiation. This study was modified by reproductive risk factors reflecting the supports the role of the MRN pathway in breast cancer level of estrogen exposure or susceptibility to estrogen development, further strengthening the suggestion that exposure, as estrogen is known to initiate breast cancer mechanisms regulating DSB repair may play a mutator development due to its metabolites causing DSB role driving breastcancer pathogenesis. (Cancer formation. Support for the hypothesis came from the Epidemiol Biomarkers Prev 2007;16(10):2024–32)

Introduction

Double-strand breaks (DSB) are extremely cytotoxic specific partner proteins. Recently, the evolutionarily DNA lesions, and cells have therefore developed an conserved Mre11/Rad50/Nbs1 (MRN) complex was extensive array of responses that lead to damage repair, implicated in ATM recruitment to DSBs (6-8). This thus preventing cell death (1-4). The nuclear protein complex is involved in the initial processing of DSBs kinase ataxia telangiectasia mutated (ATM) is regarded due to its nuclease activity and DNA binding capability, as the primary activator of this network, and the which reside in the Mre11 protein and partially depend recruitment of ATM to DNA DSBs is thought to be the on the interaction of Mre11 with Rad50, which provides critical step in its activation and function (5). However, the energy source for the MRN complex (9, 10). However, although ATM has an affinity for DNA, this recruitment in terms of the protein required for ATM recruitment, has been suggested to require and to be facilitated by it has only recently been recognized that Nbs1 recruits activated ATM to sites of DNA damage, then promotes its phosphorylation and the triggering of subsequent Received 2/7/07; revised 7/18/07; accepted 7/27/07. steps in the DNA damage response (11). These results The costs of publication of this article were defrayed in part by the payment of page show that the MRN complex functions as a DNA DSB charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact. sensor upstream of ATM signaling. However, this critical Requests for reprints: Chen-Yang Shen, Institute of Biomedical Sciences, Academia role of the complex in initiating DSBs repair does not Sinica, Taipei, 11529, Taiwan. Fax: 886-2-2782-3047. E-mail: [email protected] exclude its involvement in mechanisms downstream or Jyh-Cherng Yu, Departments of Surgery, Tri-Service General Hospital, Taipei, 11529, Taiwan. Phone: 886-2-8792-7191. E-mail: [email protected] of ATM signaling in the intra-S or G2-M cell cycle Copyright D 2007 American Association for Cancer Research. checkpoint in response to DNA damage, as suggested in doi:10.1158/1055-9965.EPI-07-0116 earlier studies (9, 10). These functional links between

Cancer Epidemiol Biomarkers Prev 2007;16(10). October 2007

ATM and the MRN complex help explain similarities in ing the effects of subtle genetic variations, such as single the clinical and cellular phenotypes associated with nucleotide polymorphisms (SNP). Furthermore, the deficiency of each of the genes encoding these proteins. use of a genetically homogenous population (i.e., the Hypomorphic mutations in Mre11 and Nbs1 result, Taiwanese population) reduces the chance of false respectively, in the human genetic instability disease, positives due to population stratification (25, 26). The ataxia-telangiectasia–like disorder, and Nijmegen break- present study included 559 female breast cancer patients age syndrome (10, 12, 13). These single-gene disorders and 1,125 healthy female controls. All subjects gave their overlap with one another and with ataxia-telangiectasia, informed consent. The recruitment of both cases and caused by ATM deficiency, all being characterized by controls and considerations regarding methodologic neurologic abnormalities, radiosensitivity, impairment of issues (such as study design, sampling scheme, and the cellular response to DSBs, and genomic instability. potential bias) have been described and addressed in The close relationship between ATM and the MRN detail previously (16, 19, 20, 25, 26). complex prompted us to hypothesize a role of the MRN Questionnaire. Two experienced research nurses were genes in determining breast cancer susceptibility. The assigned to administer a structured questionnaire to both rationale underlying this hypothesis is that carriers cases and controls. The information collected and the of either defective mutations or polymorphic variants validity of this questionnaire have been addressed and of ATM are at higher risk of developing breast cancer confirmed in our previous studies (16, 19, 20, 25, 26). (14, 15). Furthermore, we have adopted a model (i.e., the hide-then-hit hypothesis; ref. 16) to explain the lack of Genotyping. Genomic DNA was extracted from the cancer predisposition in ataxia-telangiectasia–like disor- buffy coat isolated from whole-blood samples using a der patients or no breast cancer phenotype observed in QIAamp DNA extraction kit (Qiagen, Inc.) following the manufacturer’s protocol. Nijmegen breakage syndrome patients and suggest that a 7 disparate spectrum of disease phenotypes can be Using the current data from HapMap on haplotype differently caused by mutated forms or hypomorphic/ blocks of MRN genes in the Chinese population, we polymorphic variants of the same genes. This is because, selected SNPs in each block to detect genetic variation in in contrast to common genetic diseases, cancer formation these three candidate genes and five SNPs were selected requires an extended period of time for the essential for each MRN gene. These SNPs were chosen because genomic changes to accumulate. Genetic variants or low- they are evenly distributed throughout the entire genes, penetrance alleles of DNA repair genes (such as Mre11/ a total of 15 SNPs being genotyped (Mre11: rs535801, RAD50/NBS1 examined in the present study) may rs569143, rs601391, rs684507, and rs1061945; Nbs1: therefore have a chance to escape the lethality phenotype rs1805794, rs1805790, rs709816, rs1061302, and by not triggering obvious cell cycle surveillance, and the rs1063045; Rad50: rs2252775, rs2301713, rs3798134, cells accumulate the necessary genomic instability rs2240032, and rs2244012). Because there have not been leading to cancer development. In addition, the possibil- any reports of an association between genotypic and ity of manifesting the tumorigenic phenotype depends phenotypic changes in the SNPs of the MRN genes, these not only on the joint effect of individual genes but also on selected SNPs were used as markers to reflect possible the interaction between genes and risk factors. To test linkage disequilibrium (LD) between themselves and this hypothesis, we did this investigation based (a)on different alleles of a gene of unidentified phenotypic a case control study to estimate the breast cancer risk variation. We used more than one SNP per gene to have associated with harboring putative high-risk genotypes an unbiased definition of the allelic and haplotypic of MRN genes, (b) on an examination of the joint effect statuses of each gene. of MRN genotypes and well-established risk factors of All SNPs were genotyped using a MassARRAY breast cancer in determining cancer risk, and (c)onan (SEQUENOM, Inc.). The PCR primers and extension in vivo study of the interaction between MRN and BRCA1, primers for all SNPs were designed using Spectro- encoded by the breast cancer susceptibility gene BRCA1. Designer software (SEQUENOM, Inc.). To ensure that The combination of these three lines of evidence provides the observed polymorphisms were correct and not the an essential insight into the tumorigenic contribution results of experimental variation, the results were of the MRN complex during breast cancer formation. confirmed by repeating 25% of the assays and by directly sequencing 10% of the specimens, and no inconsistent genotype was found. Materials and Methods Data Analysis. Univariate and multivariate analyses were used to determine risk factors and to establish Genotype-Based Case Control Study to Examine the background risk profiles for breast cancer, and important Association between MRN Genotypes and Breast reproductive risk factors were used as indices to estimate Cancer Risk the level of estrogen exposure or susceptibility to estrogen exposure in the subsequent analysis. Study Population. This case control study is part of an The genotypic frequency of each SNP of the individual ongoing cooperative study aimed at understanding the genes was compared between cases and controls. Differ- causes of breast cancer in Taiwan, which is characterized ences in genotypic frequency of individual SNPs between by low incidence (17), early tumor onset (18), hormone cases and controls were tested using multiple logistic dependency (19, 20), and novel genomic alterations regression models (27) with simultaneous consideration (21, 22). Because of the low incidence, which suggests an overall lower effect of common risk factors (23), and because of its homogenous genetic background (24), the Taiwanese population has certain advantages for study- 7 http://www.hapmap.org/

Cancer Epidemiol Biomarkers Prev 2007;16(10). October 2007

of known risk factors of breast cancer, and the adjusted separated by SDS-PAGE and transferred to a nitrocellu- odds ratio for the association was estimated. lose membrane, which was probed with monoclonal antibodies against polyhistidine (HIS-1, Sigma-Aldrich) Examination of Joint Effects of Genotypes and or myc (9E11, Santa Cruz) and polyclonal antiactin Reproductive Risk Factors in Determining Breast antibody (A2066, Sigma-Aldrich), followed by horserad- Cancer Risk. Our interest in identifying joint effects of ish peroxidase–conjugated secondary antibody and MRN genotypes and reproductive risk factors in deter- enhanced chemiluminescence reagent (33). mining breast cancer risk stems from the fact that exposure to estrogen, reflected by reproductive risk factors, is known to initiate breast cancer development due to estrogen metabolites causing DSB formation Results (20, 28). If MRN genes playing a role in DSB repair were Pregnancy-Related Risk Factors Are Important in important in breast tumorigenesis, the relationship Determining Breast Cancer Risk. The present study between breast cancer risk and reproductive risk factors included 559 female patients with pathologically con- would not be the same in women harboring different firmed infiltrating ductal carcinoma of the breast and MRN genotypes; this was evaluated using both joint and 1,125 healthy female controls. The risk profile of this stratified methods (29). We calculated the risk of breast series of study subjects (Table 1) was similar to that cancer associated with the combination of the putative reported in our previous breast cancer studies (16, 19, 20, high-risk genotypes of the MRN genes and a reproduc- 25, 26), and using multiple logistic regression analysis, a tive risk factor. Using b estimates from the logistic significantly increased risk was found to be conferred by regression model (30), in which we used a set of dummy a family history of breast cancer in female first-degree variables representing different combinations of gene relatives [yes versus no; adjusted odds ratio (aOR), 1.50; (i.e., whether or not harboring the putative high-risk 95% confidence interval (95% CI), 1.12-2.00]. Of the genotypes) and risk factor, we assessed the relative various reproductive risk factors, pregnancy-related risk excess risk due to harboring putative high-risk genotypes factors were consistently found to be highly associated within reproductive risk factor strata (joint method). with an increased risk. Compared with controls, cases Furthermore, the risk of breast cancer associated with had a lower frequency of a history of full-term pregnancy polymorphism of the MRN genes was compared between (FTP; no history versus having at least one FTP; aOR, women with or without a reproductive risk factor 1.51; 95% CI, 1.05-2.18) and were older at first FTP (stratified method). (z26 years versus <26 years; aOR, 1.34; 95% CI, 1.04-1.72). In vivo Pull-Down Assay to Detect an Interaction Furthermore, significant protection was conferred by a between Mre11 and BRCA1. In examining the interac- greater number of FTPs (history of z3 FTPs versus no tion between components of the MRN complex and history of FTP; aOR, 0.63; 95% CI, 0.43-0.92) or a history BRCA1, we were particularly interested in identifying of breast feeding (yes versus no; aOR, 0.70; 95% CI, an interaction between Mre11 and BRCA1, as BRCA1 0.56-0.89). The significant protection conferred by preg- has been shown to inhibit the nuclease activity of nancy against the development of breast cancer has been Mre11 (31), and it has been suggested that this suggested to be due to its causing permanent differen- inhibition is required for precise DSB repair (32). To tiation of the vulnerable breast stem cells, thus reducing this end, a pull-down assay was done. BRCA1 and susceptibility to estrogen exposure (34). These risk Mre11 were cloned, respectively, into the expression factors were therefore used in the subsequent analysis vectors pXJ-Myc (Myc epitope–tagged in the pXJ to examine the presence of a joint effect on breast cancer vector) and pcDNA3.1-His (Invitrogen), and the Myc- risk of agents causing DSBs (i.e., estrogen exposure or tagged BRCA1 or/and His-tagged Mre11 were expres- susceptibility to estrogen exposure reflected by signifi- sed in 293T cells and the cells harvested after 50 h; one cant pregnancy-related risk factors) and the MRN genes. set of cells expressing both proteins was exposed to a No association was found between cancer risk and single dose of 10 Gy radiation 48 h after transfection smoking status, radiation exposure, hormone replace- and harvested 2 h later. Protein extracts were prepared ment therapy, or dietary intake of specific kinds of foods as described previously (32), and His-tagged Mre11 was or vegetables, but obese women (body mass index, pulled-down using Ni-NTA agarose (Sigma). The >24kg/m 2) showed a significantly higher risk (aOR, 1.30; affinity purified complex and whole-cell extract were 95% CI, 1.04-1.63).

Table 1. Comparison of cases and controls by selected demographic factors and major risk factors for breast cancer from the Breast Cancer Study in Taiwan

Cases (n = 559) Controls (n = 1,125) P Age at diagnosis (y, mean F SD) 50.3 F 11.2 47.1 F 10.4<0.05 School years (y, mean F SD) 9.1 F 4.6 11.0 F 4.2 <0.05 Age at menarche (%, VÀ13 y) 32.1 35.9 0.15 Family history of breast cancer (%) 11.5 10.3 0.46 FTP ever (%) 90.1 88.5 0.20 Age at first FTP (% of >25 y) 50.451.8 0.74 Breast feeding ever (%) 52.9 49.8 0.24 Oral contraceptive use ever (%) 21.3 24.6 0.14 Hormone replacement therapy use ever (%) 23.1 23.40.86

Cancer Epidemiol Biomarkers Prev 2007;16(10). October 2007

Table 2. LD coefficients for all examined SNP pairs in the expected distribution of genotypes estimated on the the Mre11, Rad50, and Nbs1 genes basis of the observed allelic frequency of each SNP, and no statistically difference was found (i.e., none of the loci Mre11 gave a significant P value in the m2 test). This suggests SNP rs535801 rs569143 rs601391 that the SNPs examined were in Hardy-Weinberg equilibrium and that possible bias due to genotyping rs684507 0.9903 0.9915 0.9644 error was less likely (35). All SNPs in the same gene were rs601391 0.9636 0.9770 rs569143 0.9585 found to be in strong LD (P < 0.01) in both cases and controls (Table 2). This finding is consistent with results Nbs1 for SNPs in other genes in Taiwanese (16, 25, 26), because the Taiwanese population is genetically homogenous SNP rs1805794rs1805790 rs709816 rs1061302 and LD between SNPs is much stronger than in other rs1063045 0.9940 0.9904 0.9765 0.9819 populations (24). rs1061302 0.9843 0.9806 0.9695 Genotypic Polymorphism of Nbs1 and a Joint Effect rs709816 0.9813 0.9813 rs1805790 0.9916 of the MRN Genes Are Associated with Breast Cancer Risk. To explore a possible association between breast Rad50 cancer and individual polymorphisms in MRN genes, the genotypic distribution of polymorphisms of indi- SNP rs2252775 rs2301713 rs3798134rs2240032 vidual genes was compared between cases and controls rs2244012 0.9890 0.9757 0.9934 0.9798 (Table 3). For the RAD50 variant examined, it is notable rs2240032 0.9819 0.9751 0.9798 that the more frequently found allele is the one rs37981340.9935 0.9826 associated with an increased risk. Furthermore, the test rs2301713 0.9718 for differences in the distribution of haplotype frequencies between cases and controls was done with a global NOTE: The National Center for Biotechnology Information SNP cluster ID for each SNP is shown in the top row and the left column. The test using the program FASTEHPLUS with 10,000 numbers represent the LD between two SNP markers, as measured by D¶, permutations (36, 37). However, possibly because the ¶ ¶ D = D / Dmax. A higher value of D indicates a higher LD, and 1.00 MRN genes were in strong LD, the analysis based on indicates complete LD. The P values for all of these pair-wise LDs were haplotypes was similar to that based on individual <0.005. SNPs (Supplementary Table S1), and as a result, we chose the SNP which showed the most significant P value in the multivariate logistic regression analyses SNPs in MRN Genes Are in Strong Linkage to represent the allelic status of individual MRN genes Disequilibrium. Fifteen SNPs of the three MRN genes (Table 3). When the effects of breast cancer risk factors were genotyped in an initial screening of 192 cases and were simultaneously adjusted, a trend to an increased 192 controls. Of these, one (rs1061945) in Mre11 was breast cancer risk was consistently found to be infrequent (frequency of the less frequent allele, <0.01) associated with harboring one additional high-risk and, so, was not genotyped in the remainder of the allele of individual MRN genes (Table 3), although samples. The remaining 14SNPs (Table 2) were geno- only Nbs1 polymorphism (i.e., the homozygous variant typed in all cases and controls. The genotyping results genotype of Nbs1) showed a borderline significant for individual SNPs in the controls were compared with association with breast cancer development. On the

Table 3. Genotype frequencies of sequence variants of the MRN genes Mre11, Rad50, and Nbs1 in breast cancer cases and controls and the aORs in relation to breast cancer risk c b SNP and genotype* No. cases (%) No. controls (%) aOR (95% CI) aOR (95% CI) Mre11 G/C (rs569143) GG 140 (25.0) 304 (27.0) 1.00 (ref.) 1.00 (ref.) GC 286 (51.2) 592 (52.6) 1.05 (0.81-1.36) 1.00 (ref.) CC 133 (23.8) 229 (20.4) 1.31 (0.96-1.78) 1.26 (0.98-1.64) Rad50 T/G (rs2252775) TT 405 (73.5) 776 (69.0) 1.21 (0.63-2.23) 1.20 (0.94-1.52) GT 139 (24.9) 313 (27.8) 1.01 (0.51-1.99) 1.00 (ref.) GG 15 (2.6) 36 (3.2) 1.00 (ref.) 1.00 (ref.) Nbs A/G (rs1805790) AA 154(27.6) 345(30.7) 1.00 (ref.) 1.00 (ref.) AG 284(50.9) 570 (50.7) 1.08 (0.84-1.38) 1.00 (ref.) GG 120 (21.5) 210 (18.6) 1.36 (1.00-1.85) 1.29 (1.00-1.69)

Abbreviation: ref., reference group. *The National Center for Biotechnology Information SNP cluster ID for each SNP is shown in parenthesis. cThe aORs and 95%CI were estimated in a logistic regression model containing breast cancer risk factors, including age, a family history of breast cancer, a history of FTP, and the body mass index. A set of dummy variables was used to reflect the different genotypes harbored by individual cases and controls. bThe aORs and 95% CI were estimated in a logistic regression model containing breast cancer risk factors, including age, a family history of breast cancer, a history of FTP, body mass index, and the genotype of each of the three MRN genes. In these regression models, for Mre11 and Nbs1, the homozygous wild-type and heterozygous genotypes were grouped together and compared with the homozygous variant; for Rad50, the homozygous variant and the heterozygous genotypes were grouped together and compared with the homozygous wild-type genotype.

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basis of the risk profiles of the individual genotypes of Risk Associated with MRN Genes Is Modified by each MRN gene (Table 3), in the case of Mre11 and Pregnancy-Related Risk Factors. We investigated Nbs1, the homozygous wild-type and heterozygous whether estrogen exposure in conjunction with the genotypes were grouped together and compared with MRN susceptibility genotypes resulted in an increased the homozygous variant genotype, whereas in the case risk of cancer using the joint method (Fig. 1) and the of Rad50, the homozygous variant and heterozygous stratified method (Table 5). To carry out this analysis, genotypes were grouped together and compared with we first classified our women into two groups, those the homozygous wild-type genotype. After these with two or three and those with zero or one putative groupings, the Nbs1 genotype remained a significant high-risk genotypes in the three MRN genes, as such determinant for breast cancer development and was a definition would give sufficient statistical power to associated with a 1.29-fold increase in risk (95% CI, address the relevant questions. The reference group 1.00-1.69; Table 3). To comprehensively assess the consisted of women harboring 0 or 1 putative high-risk relative contribution of individual MRN genes to the genotypes and less susceptible to estrogen exposure association with breast cancer development, we did a (having a history of FTP, younger at first FTP, or a logistic regression analysis considering the effects of greater number of FTPs). Our hypothesis was supported individual genes and known risk factors of breast by the finding that, in the absence of the estrogen-related cancer. Consistent with the findings in Table 3, the risk factors, the harboring of two to three putative high- breast cancer risk associated with susceptibility geno- risk genotypes was associated with a small nonsignifi- types varied for the three genes, being more significant cant increase in risk, whereas, in the presence of any one for Nbs1 (aOR, 1.29; 95% CI, 1.00-1.69) than for Mre11 of these risk factors, the harboring of the same number of (aOR, 1.25; 95% CI, 0.97-1.62) or Rad50 (aOR, 1.21; high-risk genotypes was associated with a much greater 95% CI, 0.95-1.53). and significant combined risk of breast cancer (Fig. 1). Because individual MRN proteins act collectively by The increase in breast cancer risk in this subgroup of forming a trimeric complex, which plays multiple functions upstream and downstream of ATM in the DNA DSB repair pathway (4, 9, 10), the prediction was Table 4. Risk (aOR) of breast cancer associated with that any defect due to missense variants in individual the number of putative high-risk genotypes or with genes of this complex would act by dominantly the combination (joint effect) of two putative high- interfering with the function of the normal allele of risk genotypes of the genes encoding the DNA DSB the other partner. Thus, to determine whether a joint repair proteins of the MRN complex effect of these MRN genes was associated with breast cancer development, we examined the breast cancer MRN genes No. cases No. controls aOR (95% CI)* risk associated with the number of these putative high- (%) (%) risk genotypes, using women with all three putative Number of high-risk low-risk genotypes as the reference group. As shown genotypes in Table 4, the risk of breast cancer increased None 97 (17.4) 222 (19.7) 1.00 (ref.) significantly with the number of putative high-risk One 292 (52.3) 622 (55.3) 1.05 (0.78-1.40) Two 141 (25.3) 250 (22.2) 1.37 (0.98-1.91) genotypes (Ptrend = 0.003), one additional putative Three 28 (5.0) 31 (2.8) 2.35 (1.28-4.34) high-risk MRN genotype being associated with a 1.25- With one additional putative 1.25 (1.10-1.44) fold increase in risk (95% CI, 1.10-1.44) and the highest high-risk genotype risk (aOR, 2.35; 95% CI, 1.28-4.34) being seen in women Ptrend = 0.003 harboring the high-risk genotype of all three MRN Genotype genes. Furthermore, on the basis of the defined Mre11 Nbs1 GG.GC AA,AG 343 (61.5) 733 (65.2) 1.00 (ref.) functionsoftheMRNcomplex(4,9,10),each CC AA,AG 95 (17.0) 182 (16.2) 1.14(0.85-1.53) combination of two proteins in this complex displays GG,GC GG 82 (14.7) 163 (14.5) 1.16 (0.85-1.58) specific and independent functions required for DSB CC GG 38 (6.8) 47 (4.2) 2.01 (1.24-3.26) repair or for cellular responses to DSB lesions. Two Rad50 Nbs1 examples are that (a) the binding of Nbs1 stimulates GG,GT AA,AG 118 (21.2) 281 (25.0) 1.00 (ref.) only the endonuclease activity of Mre11, whereas the GG,GT GG 35 (6.3) 68 (6.0) 1.35 (0.83-2.19) TT AA,AG 320 (57.4) 634 (56.4) 1.22 (0.93-1.59) binding of Rad50 to Mre11 stimulates both its TT GG 85 (15.2) 142 (12.6) 1.56 (1.08-2.26) exonuclease and endonuclease activities and (b) Nbs1 Mre11 Rad50 bound to Mre11 is involved in checkpoint responses GG,GC GG,GT 123 (22.0) 274 (24.4) 1.00 (ref.) (4, 9, 10). We then dissected the complex into the CC GG,GT 31 (5.6) 75 (6.7) 0.88 (0.54-1.44) individual functional components and looked for GG,GC TT 303 (54.2) 622 (55.3) 1.07 (0.82-1.40) possible joint effects contributed by any combination CC TT 102 (18.3) 154(13.7) 1.56 (1.10-2.21) of two genes of this complex. To exclude a false *The aOR of breast cancer development associated with the combined combination effect due to an unequal contribution of effects of individual high-risk genotypes of MRN genes was estimated in individual genes, we separately estimated the risk a multivariate logistic regression model, containing age, a family history associated with harboring different putative high-risk of breast cancer, a history of FTP, body mass index, and (a) a group of genotypes using a dummy variable coding scheme (30) dummy variables to represent women harboring the four possible combinations of the different genotypes of each combination of two and women with all putative low-risk genotypes as the MRN genes, (b) a group of dummy variables to represent women reference group. The results were consistent with the harboring different numbers of putative high-risk genotypes (to presence of a joint effect, a higher risk being seen in specifically calculate the aOR for individual groups), or (c) the number women harboring high-risk genotypes of both MRN of putative high-risk genotypes (to calculate the risk associated with genes (Table 4). having one additional putative genotype and the P value for the trend).

Cancer Epidemiol Biomarkers Prev 2007;16(10). October 2007

As shown in Fig. 2, in cells expressing both proteins (lanes 4and 5), co–pull down of the BRCA1 prey with the Mre11 bait was observed. The negative controls consisted of material pulled down by Ni-NTA from nontransfected cells (lane 1) or cells expressing only one of the proteins (lanes 2 and 3). These data are consistent with the suggestion that BRCA1 and the MRN complex act together in vivo as a surveillance complex to guard genomic integrity (32). It is interesting that this interaction was DNA damage–dependent, because it was increased by irradiation in the absence of any increase in the amounts of two proteins in the extracts (lane 6). Because the pull down was done with MRE11, showing little change (lanes 4to 6 in the raw of IB anti-His in Fig. 2), such an increase would be in agreement with an increase in the quantity of BRCA1 being pulled down by MRE11.

Discussion

On the basis of a multigenic model and using both epidemiologic and experimental approaches, the pres- Figure 1. Risk (aOR) of breast cancer associated with the ent study examined the contribution to breast cancer combination of putative at-risk genotypes of the MRN genes tumorigenesis of the genes encoding the proteins in and the reproductive risk factors of a history of pregnancy, age the MRN complex, the key complex involved in DSB at first FTP, or number of FTP. The aOR of breast cancer repair (1-4). This study addressed not only the breast development associated with the combination of the number of tumorigenic risk associated with the MRN genes, but putative high-risk genotypes and reproductive risk factors was also whether there is an interaction between these estimated in a multivariate logistic regression model containing genes and reproductive risk factors in relation to breast age, a family history of breast cancer, body mass index, and a cancer risk and whether MRN proteins physically group of dummy variables to represent the four different interact with the breast cancer suppressor BRCA1. combinations of genes (number of putative high-risk geno- Our study permits a more precise evaluation of the types) and reproductive risk factor status. Nulliparous women breast cancer risk associated with MRN genes and a were not included in the analysis of the combined effect on better insight into breast tumorigenesis initiated by the cancer risk of the number of high-risk genotypes and age at DSB repair genes and how this is modified by estrogen first FTP. exposure. Consistent with our hypothesis, the findings of the present study, showing that genotypic polymorphisms of women was more than additive. These results indicate the presence of a combined effect between the MRN complex and susceptibility to estrogen exposure during Table 5. Risk (aOR) and 95% CI of breast cancer breast tumorigenesis. To further verify this finding, the development associated with harboring a higher stratified method was used, in which we examined number of putative high-risk genotypes of the genes whether the breast cancer risk associated with having a encoding the DNA DSB repair proteins of the MRN higher number of high-risk genotypes (i.e., harboring complex stratified by a history of breast feeding two or three putative high-risk genotypes) was modified by a history of breast feeding. Modification of the risk History of breast feeding effect was supported by our findings, because a signif- (no. cases/controls) icant aOR associated with a higher number of putative Yes (292 of 559) No (261 of 563) high-risk MRN genotypes or with harboring one additional putative high-risk genotype of the MRN complex No. putative high-risk was only seen in those women with no history of breast genotypes feeding (Table 5). The possibility of a difference in 0-1 1.00 (ref.) 1.00 (ref.) 2-3 1.32 (0.93-1.86) 1.51 (1.08-2.11) statistical power in the detection of cancer risk due to With one additional 1.12 (0.91-1.38) 1.37 (1.12-1.69) different sample sizes in the subsets of women can be putative high-risk excluded, because the subsets with or without a history genotype of breast feeding included a similar number of women. NOTE: The aOR of breast cancer development associated with the Mre11 Binds to BRCA1 and this Interaction Is number of putative high-risk genotypes or with one additional putative Damage-Dependent. To look for experimental evidence high-risk genotype was calculated in a multivariate logistic regression supporting a breast tumorigenic contribution of the MRN model containing age, a family history of breast cancer, body mass index, genes, we determined whether there was a physical and a group of dummy variables to represent women harboring different numbers of putative high-risk genotypes (to specifically calculate the aOR interaction between Mre11 and BRCA1. His-tagged for individual groups) or the number of putative high-risk genotypes Mre11 and Myc-tagged BRCA1 were overexpressed in (to calculate the risk associated with having one additional putative 293T cells, and a Ni-NTA pull-down assay was done. genotype).

Cancer Epidemiol Biomarkers Prev 2007;16(10). October 2007

pathway. Because the SNPs analyzed are mostly in introns, do not affect amino acid coding, and therefore probably do not directly affect protein function, the observed associations between breast cancer risk and SNPs should be interpreted as the presence of LD between these SNPs and other SNPs in exons (resulting in functional polymorphism) or in regulatory regions (affecting the expression of these genes). We attempted to use more than one SNP in these genes to assign the haplotypes and to examine haplotype effects on cancer risk, but the information generated by haplotype analysis was limited due to strong LD between SNPs in the same gene. However, because of this same reason, i.e., the presence of significant pair-wide LD in all of the SNPs, it is reasonable to expect that any increased breast cancer risk caused by unknown sequence variants spanning each MRN gene would most likely be reflected indirectly by at least one of the genotyped SNPs. In addition, it is less likely that the observed association reflects the effects of other adjacent genes, because (a) based on the newly published haplotype map of the human genome, no well-defined cancer-associated genes are located in the same haplotype blocks of the MRN genes and (b)the closest putative cancer gene MMP16 (which is suggested to be abnormally expressed in renal cell carcinoma) is 1.6 Mb 5¶ of Nbs1. Furthermore, genetic heterogeneity is Figure 2. Mre11 interacts with BRCA1 in vivo, and this less of a concern in Taiwan than in the United States (24), interaction increases when the cells are irradiated. Myc-labeled and as a result, potential bias due to population BRCA1 and/or His-labeled Mre11 were expressed in 293T cells stratification is less likely to be significant in our study as indicated by the ‘‘+’’ signs; one sample of cells expressing and the chance that the functional variants targeted by both proteins was also exposed to a single dose of 10 Gy of the same SNPs of individual MRN genes are different in radiation, then tested 2 h later. Cell lysates were incubated with cases and controls due to differences in the genetic Ni-NTA agarose and the bound material and whole-cell extracts background of the two groups is small. However, we subjected to SDS gel electrophoresis and Western blotting with recognize that the sequencing of the entire gene and anti-Myc or anti-His antibodies. Antiactin antibodies were used promoter region is the definitive approach to identifying as a loading control. The results shown are typical of those for all of the important sequence variants and that a large- three separate experiments. IB, immunoblotting. After adjusting scale evaluation of these variants and functional assess- the amounts of whole-cell extracts, the ratios of BRCA1/Mre11 ments are needed to address this question. at the pull-down lines of lines 4, 5, and 6 are 0.20, 0.19, and The multigenic approach used in this study provided a 0.49, respectively. Ni-NTA agarose beads pulled down similar unique opportunity to evaluate the relative importance amount of His-tag Mre11. of the individual MRN genes in breast cancer development and led to Nbs1 being identified as the most significant of the three. The extreme C-terminal region of Nbs1 contains a conserved ATM interaction motif, the MRN genes were jointly associated with an increased deletion of which results in loss of the ability to recruit breast cancer risk, provide evidence supporting a ATM to DSB sites (11), suggesting a critical role of Nbs1 contribution of MRN genes to breast tumorigenesis. in DSB repair. Interestingly, this C-terminal region is Interestingly, an association between a specific low- distinct from the Mre11 interaction region of Nbs1, which penetrance mutation of RAD50 and susceptibility to links Nbs1 to Mre11 (11). Phenotypically, a missense hereditary breast cancer has been reported recently, variant of Mre11 affecting the nuclease activity of Mre11 and mutation carriers show increased spontaneous does not significantly affect the ability of ATM to chromosomal aberrations in their peripheral blood T phosphorylate downstream effector proteins for DSB lymphocytes (38), consistent with the role of MRN in repair (8). In addition, although an in vitro experiment maintaining genomic stability. In a recent study which has suggested that the exonuclease activity of Mre11 is looked for mutation in the consensus coding sequences required to trim the ends of DSBs, thus facilitating DSB of 13,023 genes in breast cancer tumor tissue, Mre11 was repair, other studies have questioned whether such identified as one of the hotspots of mutation (39). activity is required in vivo for efficient DSB repair (9). Together, these findings provide essential support for On the other hand, chromosome instability, a phenotypic the biological plausibility of our results. However, in characteristic of tumorigenesis, is not observed in mouse considering whether our findings represent a true cells harboring a homozygous Rad50 mutation (10). association between the SNPs of the MRN genes and Although the strength of association, measured as the breast cancer, the most important issue is the interpre- aOR and 95% CI in epidemiologic studies, between SNPs tation of the identified association between SNPs and the of susceptibility genes and cancer risk does not neces- trait. The present study used a candidate gene approach sarily correspond to the relative importance of the based on SNPs locating in the genes of the DSB repair molecular function played by each individual gene,

Cancer Epidemiol Biomarkers Prev 2007;16(10). October 2007

these results from molecular and cellular studies and recruitment requires not only Nbs1 but also BRCA1 animal models, suggesting that Nbs1 is clearly the most and that the absence of either protein results in a failure important gene of those coding for the MRN complex to recruit activated ATM to DSB sites (44). Furthermore, during DSB repair, are still of significance. It is notable we have shown that ATM, Chk2, BRCA1, and the MRN that a recent epidemiologic study also shows that Nbs1 complex interact to determine the fidelity of DNA DSBs polymorphisms and haplotypes contribute to the etiolo- end-joining (32, 43). These recently identified pathways gy of sporadic breast cancer in young non-Hispanic are critical evidence for a breast tumorigenic role of the White women (40). MRN genes. In addition, due to its nuclease activity and Evidence about the mechanism by which estrogen DNA binding capability, the MRN complex is involved causes DNA damage, thus initiating breast cancer in the initial processing of DSBs (45). These activities development, is provided not only by molecular and reside in Mre11, which is required to trim the ends of cellular studies (28), but also by epidemiologic obser- DSBs, thus facilitating DSB repair, especially during vations (19, 20). The suggested mechanism is that the error-free mechanism of homologous recombination estrogen metabolites (i.e., CE-Qs) bind to DNA, leading by producing the overhanging DSB ends required for to the formation of depurinating adducts and resulting efficient homologous recombination (3, 4). On the other in DSB formation. On the basis of this evidence, we hand, because BRCA1 inhibits the nuclease activity of examined the breast tumorigenic role of the MRN genes Mre11 (31) and Chk2 phosphorylates BRCA1, leading to by exploring whether breast cancer risk was linked to a inhibition of MRN complex foci formation (46), it has joint effect of MRN genotypes and pregnancy-related been suggested that this inhibition is required for precise risk factors. If these susceptibility genes were associated end-joining because exonuclease digestion would result with breast cancer development via the hypothesized in the loss of nucleotides, leading to imprecise end- mechanism involving the regulation of DSB repair, the joining if these digested ends were rejoined directly (32). relationship between cancer risk and susceptibility Thus, in the present study, our finding of a damage- genotypes would be expected to be more significant dependent interaction between BRCA1 and Mre11 is in in that subset of women who were expected to be more line with this hypothesized model. To further confirm susceptible to estrogen, indicated by no history of this interaction is of functional importance, in our pregnancy or breast feeding, an older age at first FTP, ongoing experiment, we have identified interaction or fewer FTPs, as these risk factors are an indicator domains of BRCA1 and Mre11 and have done functional of increased susceptibility to estrogen exposure (41). assays to examine phenotypic changes in mutant Mre11 The findings are consistent with these expectations, (unpublished observations). All these findings of phys- yielding additional support for involvement of the ical and functional interactions between individual MRN MRN genes in breast cancer development. More components and proteins encoded by well-documented importantly, these observations may help explain the breast cancer susceptibility genes provide essential issue of tissue specificity and why the DSB repair support for a tumorigenic contribution of MRN genes mechanisms are of particular importance in the devel- during breast cancer formation. opment of breast cancer, as the risk factors of increased The great interest in defining the breast tumorigenic estrogen exposure or increased susceptibility to estrogen role of the genes involved in DSB repair, such as the exposure presumably reflect the extent of DSB forma- MRN genes, arose from an attempt to confirm the tion or the degree of susceptibility to DSB formation. ‘‘mutator phenotype’’ hypothesis (47). Given that Consequently, breast cells that have lost DSB-related tumorigenesis is a multistep process, resulting from a checkpoint/repair due to the harboring of at-risk series of genomic alterations, which lead to the genotypes have a growth advantage over DSB check- progressive disordering of the normal mechanisms point/repair-proficient cells and are selected for by the controlling the growth, differentiation, and death of microenvironment imposed by estrogen-related risk the cell, this hypothesis suggests that, to account for the factors, resulting in an increased risk of developing high frequency of genomic alterations required for breast cancer. Interestingly, our inference about the tumor progression, the genomes of cancer cells are interaction between DSB repair genes and estrogen unstable and that defective mutators cause these exposure is supported by genetic evidence that repro- genomic instabilities. The fact that the familial breast ductive history might influence cancer risk in women cancer susceptibility genes BRCA1 and BRCA2 are with BRCA1 or BRCA2 mutation (42). involved in the homologous recombination pathway Our attempt to identify an interaction between the for DNA DSB repair (48) and our previous finding that MRN complex and BRCA1 was based on the extension of the extent of DSB-initiated chromosomal instability in how we view the etiology of tumorigenesis from single- tumors increases significantly, as tumors progress to gene mechanisms to multigenetic or to etiologic pathway- poorer grades or later stages (21) support the idea that wide networks (43). Consequently, the consideration breast cancer pathogenesis is driven by DSB-initiated of whether there is a causal link between a putative chromosomal instability, and we hypothesize that the cancer-associated gene and tumor development might be mechanisms regulating DSB repair may play a mutator extended to whole tumorigenic networks. Given this role. In our recent studies (16, 25, 26, 49), we have revised concept, it has become essential to explore the identified specific molecular DSB repair mechanisms, presence of functional interactions between the MRN functional aberrations of which are related to an complex and breast cancer susceptibility genes, including increased risk of breast cancer and advanced patho- ATM, Chk2, and BRCA1. In addition to the upstream logic/clinical manifestations. The results of the present role of the MRN complex in recruiting ATM (6-8, 11), thus study provide additional evidence supporting the initiating a series of responses to repair DSBs as mutator role of DSB repair mechanisms during breast mentioned above, there is recent evidence that this cancer formation.

Cancer Epidemiol Biomarkers Prev 2007;16(10). October 2007

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